LAB INVESTIGATION OF ANAEMIAS

MADHURA SHEKATKAR
GUIDED BY: DR. SUPRIYA KHEUR AND DR. MAMATHA REDDY
DEPARTMENT OF ORAL PATHOLOGY
DATE: 21/09/21
 ANAEMIA

 Anaemia is defined as a reduction in the concentration of circulating haemoglobin below the lower

limit of the normal range for the age and sex of the individual.

 Anaemia is one of the most common clinical conditions encountered. It can vary from a mild,

clinically quiescent condition to a serious, incapacitating disability. Its causes vary from a modest
mismatch between the iron requirements and intakes of a pregnant woman to the only overt sign of
advanced malignant or other systemic disease.
 CLASSIFICATION OF ANAEMIAS

 Morphological
 Etiological
 Based on reticulocyte response.
 GRADING OF ANAEMIA

 Mild : Hb from lower limit of normal range to 10.0 g/dl

 Moderate : 10.0 to 7.0 g/dl

 Severe : < 7.0 g/dl

NORMAL PROCESS
 ERYTHROPOIESIS

 Erythropoiesis is production of mature erythrocytes of the peripheral blood which takes place in the
bone marrow from morphologically unrecognizable HSC.
 Red cell production is influenced by growth factors and hormones, notably erythropoietin.
 ERYTHROPOIETIN

 Erythropoietic activity in the body is regulated by erythropoietin, which is produced in response to

anoxia.
 The principal site of erythropoietin production is the kidney though there is evidence of its extra-renal
production in certain unusual circumstances.
 Its levels are, therefore, lowered in chronic renal diseases, while a case of renal cell carcinoma may be
associated with its increased production and erythrocytosis.
 Erythropoietin acts on the marrow at the various stages of morphologically unidentifiable as well as
identifiable erythroid precursors.
 SIGNIFICANCE

 There is an increased production of erythropoietin in most types of anaemias. However, in anaemia of

chronic diseases (e.g. in infections and neoplastic conditions) there is no such enhancement of
erythropoietin.
 In polycythemia rubra vera, there is erythrocytosis but depressed production of erythropoietin. This is
because of an abnormality of HSC class which is not under erythropoietin control.
 Besides erythropoietin, androgens and thyroxine also appear to be involved in the red cell production.
 THE ERYTHROID SERIES

Erythroid series are a well-defined and readily recognizable lineage of nucleated red cells normally confined to the
marrow. These are as under:

The erythroid series: There is progressive condensation of the nuclear chromatin which is eventually extruded from the cell
at the late erythroblast stage. The cytoplasm contains progressively less RNA and more haemoglobin.
 THE RED CELL

 The mature erythrocytes of the human peripheral blood are non-nucleated cells and lack the usual cell
organelles.
 The normal human erythrocyte is a biconcave disc, 7.2 µm in diameter, and has a thickness of 2.4 µm
at the periphery and 1 µm in the center.
 The biconcave shape renders the red cells quite flexible so that they can pass through capillaries whose
minimum diameter is 3.5 µm.
 More than 90% of the weight of erythrocyte consists of haemoglobin.
 The lifespan of red cells is 120 + 30 days.
 HAEMOGLOBIN
Haemoglobin consists of a basic protein, globin, and the iron-porphyrin complex, haem. The molecular weight of
haemoglobin is 68,000. Normal adult haemoglobin (HbA) constitutes 96-98% of the total haemoglobin content and consists
of four polypeptide chains, a2b2. Synthesis of haem occurs largely in the mitochondria by a series of biochemical reactions.
RED CELL FUNCTIONS

1) Oxygen carrying
2) CO2 transport
 NORMAL VALUES AND RED CELL INDICES

 Range of normal red cell count in health is 5.5 ± 1.0 × 1012/L in men and 4.8 ± 1.0 × 1012/L in

women.

 The packed cell volume (PCV) or hematocrit is the volume of erythrocytes per liter of whole blood

indicating the proportion of plasma and red cells and ranges 0.47 ± 0.07 L/L (40-54%) in men and 0.42
± 0.05 L/L (37-47%) in women.

 The haemoglobin content in health is 15.5 ± 2.5 g/dl (13-18 g/dl) in men and 14.0 ± 2.5 g/dl (11.5-16.5

g/dl) in women.
 Based on these normal values, a series of absolute values or red cell indices can be derived which have
diagnostic importance. These are as under:
Since MCHC is independent of red cell count and
size, it is considered to be of greater clinical
significance as compared to other absolute values.
It is low in iron deficiency anaemia but is usually
normal in macrocytic anaemia.
4. Red cell distribution width (RDW):
RDW is an assessment of varying volume of red
cells based on size of red cells. For example,
fragmented red cells have a tiny size while the
macrocytes and reticulocytes have large size.
 RED CELL INDICES

 An alternative method to diagnose and detect the severity of anaemia is by measuring the red cell
indices:
 In iron deficiency and thalassaemia, MCV, MCH and MCHC are reduced.
 In early stage of iron deficiency, RDW is increased while in thalassaemia trait RDW is normal (with
low MCV) and can be distinguished from iron deficiency.
 In anaemia due to acute blood loss and haemolytic anaemias, MCV, MCH and MCHC are all within
normal limits.
 In megaloblastic anaemias, MCV is raised above the normal range.
 INTRODUCTION

Laboratory investigations play an essential part in diagnosing anaemia, establishing its etiology and
determining and monitoring its appropriate treatment. 
 GENERAL SCHEME OF INVESTIGATIONS OF ANAEMIA

 After obtaining the full medical history pertaining to different general and specific signs and symptoms,

the patient is examined for evidence of anaemia.

 Special emphasis is placed on color of the skin, conjunctivae, sclerae and nails.

 Changes in the retina, atrophy of the papillae of the tongue, rectal examination for evidence of bleeding,

and presence of hepatomegaly, splenomegaly, lymphadenopathy and bony tenderness are looked for.

 In order to confirm or deny the presence of anaemia, its type and its cause, the following plan of

investigations is generally followed, of which complete blood counts (CBC) with reticulocyte count is
the basic test.
 HAEMOGLOBIN ESTIMATION

 The first and foremost investigation in any suspected case of anaemia is to carry out a haemoglobin

estimation.

 Several methods are available but most reliable and accurate is the cyanmethaemoglobin (HiCN)

method employing Drab kin's solution and a spectrophotometer.

 If the haemoglobin value is below the lower limit of the normal range for particular age and sex, the

patient is said to be anaemic.

 In pregnancy, there is hemodilution and, therefore, the lower limit in normal pregnant women is less

(10.5 g/dl) than in the non-pregnant state.

 PERIPHERAL BLOOD FILM EXAMINATION

 Examination of a peripheral blood film for morphologic features after staining it with the

Romanowsky dyes (e.g. Leishman’s stain, May-Grünwald-Giemsa’s stain, Jenner-Giemsa’s stain,

Wright’s stain etc).

 The blood smear is evaluated in an area where there is neither Rouleaux formation nor so thin as to

cause red cell distortion.

 VARIATION IN SIZE (ANISOCYTOSIS)

 There is slight variation in diameter of the red cells from 6.7-7.7 µm (mean value 7.2 µm).

 Increased variation in size of the red cell is termed anisocytosis.

 Anisocytosis may be due to the presence of cells larger than normal (macrocytosis) or cells smaller than normal (microcytosis).

 Sometimes both microcytosis and macrocytosis are present (dimorphic).

 i) Macrocytes are classically found in megaloblastic anaemia; other causes are aplastic anaemia, other dyserythropoietic

anaemias, chronic liver disease and in conditions with increased erythropoiesis.

 ii) Microcytes are present in iron deficiency anaemia, thalassaemia and spherocytosis.

 They may also result from fragmentation of erythrocytes such as in haemolytic anaemia.
 VARIATION IN SHAPE (POIKILOCYTOSIS)

 Increased variation in shape of the red cells is termed poikilocytosis.

 The nature of the abnormal shape determines the cause of anaemia.

 Poikilocytes are produced in various types of abnormal erythropoiesis e.g. in megaloblastic anaemia,

iron deficiency anaemia, thalassaemia, myelosclerosis and microangiopathic haemolytic anaemia.

 INADEQUATE HAEMOGLOBIN FORMATION (HYPOCHROMASIA)

 The intensity of pink staining of haemoglobin in a Romanowsky-stained blood smear gradually

decreases from the periphery to the centre of the cell.

 Increased central pallor is referred to as hypochromasia. It may develop either from lowered

haemoglobin content (e.g. in iron deficiency anaemia, chronic infections), or due to thinness of the red
cells (e.g. in thalassaemia, sideroblastic anaemia).

 Unusually deep pink staining of the red cells due to increased haemoglobin concentration is termed

hyperchromasia and may be found in megaloblastic anaemia, spherocytosis and in neonatal blood.
 COMPENSATORY ERYTHROPOIESIS

 Polychromasia is defined as the red cells having more than one type of color. Polychromatic red cells

are slightly larger, generally stained bluish-grey and represent reticulocytes and, thus, correlate well
with reticulocyte count.

 Erythroblastaemia is the presence of nucleated red cells in the peripheral blood film.

 Punctate basophilia or basophilic stippling is diffuse and uniform basophilic granularity in the cell

which does not stain positively with Perls’ reaction (in contrast to Pappenheimer bodies which stain
positively).

 Howell-Jolly bodies are purple nuclear remnants, usually found singly, and are larger than basophilic

stippling. They are present in megaloblastic anaemia and after splenectomy.

 MISCELLANEOUS CHANGES

 Spherocytosis.
 Schistocytosis
 Irregularly contracted red cells
 Leptocytosis
 Sickle cells or drepanocytes
 Crenated red cells
 Acanthocytosis
 Burr cells
 Stomatocytosis
 Ovalocytosis or elliptocytosis
 LEUCOCYTE AND PLATELET COUNT

 Measurement of leucocyte and platelet count helps to distinguish pure anaemia from pancytopenia in

which red cells, granulocytes and platelets are all reduced.

 In anaemias due to haemolysis or haemorrhage, the neutrophil count and platelet counts are often

elevated.

 In infections and leukaemias, the leucocyte counts are high and immature leucocytes appear in the

blood.
 RETICULOCYTE COUNT

 Reticulocyte count (normal 0.5-2.5%) is done in each case of anaemia to assess the marrow

erythropoietic activity.

 In acute haemorrhage and in haemolysis, the reticulocyte response is indicative of impaired marrow

function.
 ERYTHROCYTE SEDIMENTATION RATE

 The ESR is a non-specific test used as a screening test for anaemia.

 It usually gives a clue to the underlying organic disease but anaemia itself may also cause rise in the

ESR.
 BONE MARROW EXAMINATION

 Bone marrow examination may be performed by two methods—aspiration and trephine biopsy.

 Bone marrow aspiration is done in cases where the cause for anaemia is not obvious.
 BONE MARROW ASPIRATION

 The marrow film provides assessment of cellularity, details of developing blood cells (i.e. normoblastic or

megaloblastic, myeloid, lymphoid, macrophages and megakaryocytic), ratio between erythroid and myeloid cells,
storage diseases, and for the presence of cells foreign to the marrow such as secondary carcinoma, granulomatous
conditions, fungi (e.g. histoplasmosis) and parasites (e.g. malaria, leishmaniasis, trypanosomiasis).

 Estimation of the proportion of cellular components in the marrow, however, can be provided by doing a

differential count of at least 500 cells.

 In some conditions, the marrow cells can be used for more detailed special tests such as cytogenetics,

microbiological culture, biochemical analysis, and immunological and cytological markers.

 TREPHINE BIOPSY

 Trephine biopsy is performed by a simple Jamshidi trephine needle by which a core of tissue from periosteum to

bone marrow cavity is obtained.

 The tissue is then fixed, soft decalcified and processed for histological sections and stained with haematoxylin and

eosin and for reticulin.

 Trephine biopsy is useful over aspiration since it provides an excellent view of the overall marrow architecture,

cellularity, and presence or absence of infiltrates, but is less valuable than aspiration as far as individual cell
morphology is concerned.
Based on the red cell size, haemoglobin content and red cell indices, anaemias are classified into 3 types:
1. Microcytic, hypochromic MCV, MCH, MCHC are all reduced e.g. in iron deficiency anaemia and in
certain non-iron deficient anaemias (sideroblastic anaemia, thalassaemia, anaemia of chronic disorders).
2. Normocytic, normochromic MCV, MCH, MCHC are all normal e.g. after acute blood loss, haemolytic
anaemias, bone marrow failure, anaemia of chronic disorders.
3. Macrocytic MCV is raised e.g. in megaloblastic anaemia due to deficiency of vitamin B12 or folic acid.
HYPOCHROMIC ANAEMIAS
 IRON DEFICIENCY ANAEMIA

 The development of anaemia progresses in 3 stages:

 Firstly, storage iron depletion occurs during which iron reserves are lost without compromise of the

iron supply for erythropoiesis.

 The next stage is iron deficient erythropoiesis during which the erythroid iron supply is reduced

without the development of anaemia.

 The final stage is the development of frank iron deficiency anaemia when the red cells become
microcytic and hypochromic.
 LAB FINDINGS

 Usually mild to moderate but occasionally it may be marked (haemoglobin less than 6 g/dl) due to persistent and

severe blood loss.

 Haemoglobin: The essential feature is a fall in haemoglobin concentration up to a variable degree.

 The red cells in the blood film are hypochromic and microcytic, and there is anisocytosis and poikilocytosis.

Hypochromia generally precedes microcytosis.

 The reticulocyte count is normal or reduced but may be slightly raised (2-5%) in cases after haemorrhage.

 The red cell indices reveal a diminished MCV (below 50 fl), diminished MCH (below 15 pg), and diminished MCHC

(below 20 g/dl).

 The total and differential white cell counts are usually normal.

 Platelet count is usually normal but may be slightly to moderately raised in patients who have had recent bleeding.
BONE MARROW FINDINGS:
i) The marrow cellularity is increased due to erythroid hyperplasia (myeloid-erythroid ratio decreased).
ii) There is normoblastic erythropoiesis with predominance of small polychromatic normoblasts (micro-normoblasts).
iii) Other cells Myeloid, lymphoid and megakaryocytic cells are normal in number and morphology.
iv) Marrow iron staining (Prussian blue reaction) on bone marrow aspirate smear shows deficient reticuloendothelial iron
stores and absence of siderotic iron granules from developing normoblasts.
BIOCHEMICAL FINDINGS:
v) The serum iron level is low (normal 40-140 µg/dl); it is often under 50 µg/dl. When serum iron falls below 15 µg/dl,
marrow iron stores are absent.
vi) Total iron binding capacity (TIBC) is high (normal 250- 450 µg/dl) and rises to give less than 10% saturation (normal
33%).
vii) Serum ferritin is very low (normal 30-250 ng/ml) indicating poor tissue iron stores.
viii)Red cell protoporphyrin is very low (normal 20-40 µg/dl) as a result of insufficient iron supply to form haem.
ix) Serum transferrin receptor protein which is normally present on developing erythroid cells and reflects total red cell
 SIDEROBLASTIC ANAEMIA

 The sideroblastic anaemias comprise a

group of disorders of diverse etiology in

which the nucleated erythroid precursors
in the bone marrow, show characteristic
‘ringed sideroblasts.

 Siderocytes and sideroblasts are

erythrocytes and normoblasts
respectively which contain cytoplasmic
granules of iron.
 LABORATORY FINDINGS

 There is generally moderate to severe degree of anaemia.

 The blood picture shows hypochromic anaemia which may be microcytic, or there may be some normocytic red

cells as well (dimorphic).

 Absolute values (MCV, MCH and MCHC) are reduced in hereditary type but MCV is often raised in acquired type.

 Bone marrow examination shows erythroid hyperplasia with usually macro-normoblastic erythropoiesis.

 Marrow iron stores are raised and pathognomonic ring sideroblasts are present.

 Serum ferritin levels are raised.

 Serum iron is usually raised with almost complete saturation of TIBC.

 There is increased iron deposition in the tissue.

ANAEMIA OF CHRONIC DISORDERS
 LABORATORY FINDINGS
 Anaemia is generally mild to moderate. A haemoglobin value of less than 8 g/dl suggests the presence of
additional contributory factors.
 The type of anaemia in these cases is generally normocytic normochromic but may have slight microcytosis and
hypochromia.
 MCHC is slightly low.
 The reticulocyte count is generally low.
 Measurement of erythrocyte survival generally reveals mild to moderate shortening of their lifespan.
 Bone marrow examination of the marrow generally reveals normal erythroid maturation.
 Serum iron is characteristically reduced in this group of anaemias while TIBC is low to-normal.
 Serum ferritin levels are increased in these patients and is the most distinguishing feature between true iron-
deficiency anaemia and iron-deficient erythropoiesis in anaemia of chronic diseases.
 In addition, certain other plasma proteins called ‘phase reactants’ are raised in patients with chronic inflammation,
probably under the stimulus of interleukin-1 released by activated macrophages.
MEGALOBLASTIC ANAEMIAS—VITAMIN B12 AND
FOLATE DEFICIENCY
 LABORATORY FINDINGS

 The investigations of a suspected case of megaloblastic anaemia are aimed at 2 aspects:

 General laboratory investigations of anaemia which include blood picture, red cell indices, bone marrow findings,
and biochemical tests.
 Special tests to establish the cause of megaloblastic anaemia as to know whether it is due to deficiency of vitamin
B12 or folate.
 GENERAL LABORATORY FINDINGS

 Haemoglobin estimation reveals values below the normal range.

 Red blood cell morphology in a blood film shows the characteristic macrocytosis.
 In addition, the blood smear demonstrates marked anisocytosis, poikilocytosis and presence of macro-
ovalocytes.
 Basophilic stippling and occasional normoblast may also be seen
 The reticulocyte count is generally low to normal in untreated cases.
 The red cell indices reveal an elevated MCV (above 120 fl).
 The total white blood cell count may be reduced.
 An occasional myelocyte may also be seen.
 Platelet count may be moderately reduced in severely anaemic patients. Bizarre forms of platelets may
be seen.
 The marrow is hypercellular with a decreased myeloid-erythroid ratio.

 There is erythroid hyperplasia due to characteristic megaloblastic erythropoiesis.

 Megaloblasts are abnormal, large, nucleated erythroid precursors, having nuclear-cytoplasmic asynchrony i.e. the nuclei are

less mature than the development of cytoplasm.

 The nuclei are large, having fine, sieve-like and open chromatin that stains lightly, while the hemoglobinization of the

cytoplasm proceeds normally or at a faster rate i.e. nuclear maturation lags behind that of cytoplasm (compared from iron
deficiency anaemia in which cytoplasmic maturation lags behind.

 Megaloblasts with abnormal mitoses may be seen.

 Granulocyte precursors are also affected to some extent.

 Giant forms of metamyelocytes and band cells may be present in the marrow. Prussian blue staining for iron in the marrow

shows an increase in the number and size of iron granules in the erythroid precursors.

 Marrow cells may show variety of random chromosomal abnormalities such as chromosome breaks, centromere spreading

etc.
BIOCHEMICAL FINDINGS:
i) There is rise in serum unconjugated bilirubin and LDH as a result of ineffective
erythropoiesis causing marrow cell breakdown.
ii) The serum iron and ferritin may be normal or elevated.
SPECIAL TESTS FOR CAUSE OF SPECIFIC
DEFICIENCY
 TESTS FOR VITAMIN B12 DEFICIENCY
 SERUM VITAMIN B12 ASSAY:

 Microbiological assay: In this test, the serum sample to be assayed is added to a medium containing all other

essential growth factors required for a vitamin B12-dependent microorganism. The medium along with
microorganism is incubated and the amount of vitamin B12 is determined turbimetrically which is then compared
with the growth produced by a known amount of vitamin B12. Several organisms have been used for this test such
as Euglena gracilis, Lactobacillus leichmannii, Escherichia coli and Ochromonas malhamensis. E. gracilis is,
however, considered more sensitive and accurate. The addition of antibiotics to the test interferes with the growth
and yields false low result.

 Radioassay of serum B12 by radioisotope dilution (RID) and radioimmunoassay (RIA) have been developed. These

tests are more sensitive and have the advantage over microbiologic assays in that they are simpler and more rapid,
and the results are unaffected by antibiotics and other drugs which may affect the living organisms.
 SCHILLING TEST (24-HOUR URINARY EXCRETION TEST)

 Schilling test is done to detect vitamin B12 deficiency as well as to distinguish and detect lack of IF and
malabsorption syndrome.
 The results of test also depend upon good renal function and proper urinary collection.
 The test is performed in 3 stages as under:
 Stage I: Without IF a) In normal individuals, 24-hour urinary excretion is >10% of the oral dose of ‘hot’ B12.

b) Patients with IF deficiency excrete lower quantity of ‘hot’ B12 which is further confirmed by repeating the test
as in stage II given below.
 Stage II: With IF
 Patients with pernicious anaemia have abnormal test even after treatment with vitamin B12 due to IF
deficiency.
 Stage III: In conditions causing malabsorption, the test is repeated after a course of treatment with
antibiotics or anti-inflammatory drugs.
 SERUM ENZYME LEVELS:
 Besides Schilling test, another way of distinguishing whether megaloblastic anaemia is due to
cobalamine or folate is by serum determination of methylmalonic acid and homocysteine by
sophisticated enzymatic assays.
 Both are elevated in cobalamine deficiency, while in folate deficiency there is only elevation of
homocysteine and not of methylmalonic acid.
 TESTS FOR FOLATE DEFICIENCY

 Folic acid is required for conversion of formiminoglutamic acid (FIGLU) to glutamic acid in the
catabolism of histidine. Thus, on oral administration of histidine, urinary excretion of FIGLU is increased
if folate deficiency is present.
 The folate in serum can be estimated by 2 methods—microbiological assay and radio assay.

i) Microbiological assay: This test is based on the principle that the serum folate acid activity is mainly due
to the presence of a folic acid co-enzyme, 5-methyl THF, and that this compound is required for growth
of the microorganism, Lactobacillus casei. The growth of L. casei is inhibited by addition of antibiotics.
ii) Radio assay: The principle and method of radio assay by radioisotope dilution (RID) test are similar to
that for serum B12 assay. The test employs labelled pteroylglutamic acid or methyl-THF. Commercial
kits are available which permit simultaneous assay of both vitamin B12 and folate.
 RED CELL FOLATE ASSAY:
 Red cells contain 20-50 times more folate than the serum; thus red cell folate assay is more reliable
indicator of tissue stores of folate than serum folate assay.
 Microbiological radio assay and protein binding assay methods can be used for estimation of red cell
folate.
 Red cell folate values are decreased in patients with megaloblastic anaemia as well as in patients with
pernicious anaemia.
HAEMOGLOBINOPATHIES
LAB INVESTIGATIONS OF ANAEMIA
PART II
STRUCTURALLY ABNORMAL HAEMOGLOBINS
SICKLE SYNDROMES
 HETEROZYGOUS STATE: SICKLE CELL TRAIT

 These patients have no anaemia and have normal appearance of red cells. But in
hypoxic crisis, sickle cell crises develop.
 The diagnosis is made by 2 tests:
 Demonstration of sickling done under condition of reduced oxygen tension by an oxygen
consuming reagent, sodium metabisulfite.
 Haemoglobin electrophoresis reveals 35-40% of the total haemoglobin as HbS.
 HOMOZYGOUS STATE: SICKLE CELL ANAEMIA

 Moderate to severe anaemia (haemoglobin concentration 6-9 g/dl).

 The blood film shows sickle cells and target cells and features of splenic atrophy such as presence of
Howell-Jolly bodies.
 A positive sickling test with a reducing substance such as sodium metabisulfite.
 Haemoglobin electrophoresis shows no normal HbA but shows predominance of HbS and 2-20% HbF
REDUCED GLOBIN CHAIN SYNTHESIS:
THALASSAEMIAS
 DEFINITION

 In a-thalassaemia major, the obvious cause of anaemia is the inability to synthesize adult haemoglobin,
while in a-thalassaemia trait there is reduced production of normal adult haemoglobin.
 In b-thalassaemia major, the most important cause of anaemia is premature red cell destruction brought
about by erythrocyte membrane damage caused by the precipitated a-globin chains.
 Other contributory factors are: shortened red cell lifespan, ineffective erythropoiesis, and
haemodilution due to increased plasma volume.
 A deficiency of b-globin chains in b-thalassaemia leads to large excess of a-chains within the
developing red cells.
 Part of these excessive a-chains are removed by pairing with g-globin chains as HbF, while the
remainder unaccompanied a-chains precipitate rapidly within the red cell as Heinz bodies
 ALPHA-THALASSAEMIA

Alpha-thalassaemias are classified into 4 types:

1. Four a-gene deletion: Hb Bart’s hydrops foetalis.
2. Three a-gene deletion: HbH disease.
3. Two a-gene deletion: a-thalassaemia trait.
4. One a-gene deletion: a-thalassaemia trait (carrier)
 HB BART’S HYDROPS FOETALIS

 Severe anaemia (haemoglobin below 6 g/dl).

 Blood film show marked aniso-poikilocytosis, hypochromia, microcytosis, polychromasia, basophilic
stippling, numerous normoblasts and target cells.
 Reticulocyte count is high.
 Serum bilirubin level is elevated.
 Haemoglobin electrophoresis shows 80-90% Hb-Bart’s and a small amount of Hb-H and Hb-Portland
but no HbA, HbA2 or HbF.
 HBH DISEASE

 Moderate anaemia (haemoglobin 8-9 g/dl).

 Blood film shows severe microcytosis, hypochromia, basophilic stippling, target cells and normoblasts.
 Mild reticulocytosis.
 HbH inclusions as Heinz bodies can be demonstrated in mature red cells with brilliant cresyl blue
stain.
 Haemoglobin electrophoresis shows 2-4% HbH and the remainder consists of HbA, HbA2 and HbF.
 A-THALASSAEMIA TRAIT

 Haemoglobin level normal or mildly reduced.

 Blood film shows microcytic and hypochromic red cell morphology but no evidence of haemolysis or
anaemia.
 MCV, MCH and MCHC may be slightly reduced.
 Haemoglobin electrophoresis reveals small amount of Hb-Bart’s in neonatal period (1-2% in a-
thalassaemia 2 and 5-6% in a-thalassaemia 1) which gradually disappears by adult life. HbA2 is either
normal or slightly decreased (contrary to the elevated HBA2 levels in b-thalassaemia trait).
 B-THALASSAEMIAS
 Anaemia, usually severe.
 Blood film shows severe microcytic hypochromic red cell morphology, marked anisopoikilocytosis, basophilic stippling, presence of many
target cells, tear drop cells and normoblasts.
 Serum bilirubin (unconjugated) is generally raised.
 Reticulocytosis is generally present.
 MCV, MCH and MCHC are significantly reduced.
 WBC count is often raised with some shift to left of the neutrophil series, with presence of some myelocytes and metamyelocytes.
 Platelet count is usually normal but may be reduced in patients with massive splenomegaly.
 Osmotic fragility characteristically reveals increased resistance to saline haemolysis i.e. decreased osmotic fragility.
 Haemoglobin electrophoresis shows presence of increased amounts of HbF, increased amount of HbA2, and almost complete absence or
presence of variable amounts of HbA.
 Bone marrow aspirate examination shows normoblastic erythroid hyperplasia with predominance of intermediate and late normoblasts
which are generally smaller in size than normal.
 Iron staining demonstrates siderotic granules in the cytoplasm of normoblasts, increased reticuloendothelial iron but ring sideroblasts are
only occasionally seen.
 B-THALASSAEMIA MINOR

 Mild anaemia; mean haemoglobin level is about 15% lower than in normal person for the age and sex.
 Blood film shows mild anisopoikilocytosis, microcytosis and hypochromia, occasional target cells and
basophilic stippling.
 Serum bilirubin may be normal or slightly raised.
 Mild reticulocytosis is often present.
 MCV, MCH and MCHC may be slightly reduced.
 Osmotic fragility test shows increased resistance to haemolysis i.e. decreased osmotic fragility.
 Haemoglobin electrophoresis is confirmatory for the diagnosis and shows about two-fold increase in
HbA2 and a slight elevation in HbF (2-3%).
ANAEMIA OF BLOOD LOSS
 ACUTE BLOOD LOSS

i) Normocytic and normochromic anaemia

ii) Low haematocrit
iii) Increased reticulocyte count in peripheral blood (10-15% after one week) reflecting
accelerated marrow erythropoiesis.
 APLASTIC ANAEMIA
 Anaemia Haemoglobin levels are moderately reduced. The blood picture generally shows normocytic
normochromic anaemia but sometimes macrocytosis may be present. The reticulocyte count is reduced
or zero.
 The absolute granulocyte count is particularly low (below 1500/µl) with relative lymphocytosis. The
neutrophils are morphologically normal but their alkaline phosphatase score is high.
 Thrombocytopenia Platelet count is always reduced.
 A bone marrow aspirate may yield a ‘dry tap’.
 A trephine biopsy is generally essential for making the diagnosis which reveals patchy cellular areas in
a hypocellular or aplastic marrow due to replacement by fat. There is usually a severe depression of
myeloid cells, megakaryocytes and erythroid cells so that the marrow chiefly consists of lymphocytes
and plasma cells.
 Haematopoietic stem cells bearing CD34 marker are markedly reduced or absent.
HAEMOLYTIC ANAEMIAS AND ANAEMIA DUE TO
BLOOD LOSS
 LABORATORY EVALUATION OF HAEMOLYSIS

Tests of Increased Red Cell Breakdown

1. Serum bilirubin—unconjugated (indirect) bilirubin is raised.
2. Urine urobilinogen is raised but there is no bilirubinuria.
3. Faecal stercobilinogen is raised.
4. Serum haptoglobin (a-globulin binding protein) is reduced or absent.
5. Plasma lactic dehydrogenase is raised.
6. Evidences of intravascular haemolysis in the form of haemoglobinaemia, haemoglobinuria,
methemoglobinemia and haemosiderinuria
Tests of Increased Red Cell Production:
1. Reticulocyte count reveals reticulocytosis which is generally early and is hence most useful initial test
of marrow erythroid hyperplasia.
2. Routine blood film shows macrocytosis, polychromasia and presence of normoblasts.
3. Bone marrow shows erythroid hyperplasia with usually raised iron stores.
4. X-ray of bones shows evidence of expansion of marrow space, especially in tubular bones and skull.
Tests of Damage to Red Cells:
1. Routine blood film shows a variety of abnormal morphological appearances of red cells
2. Osmotic fragility is increased or decreased.
3. Auto-haemolysis test with or without addition of glucose.
4. Coombs’ antiglobulin test.
5. Electrophoresis for abnormal haemoglobin.
6. Estimation of HbA2.
7. Estimation of HbF.
8. Tests for sickling.
9. Screening test for G6PD deficiency and other enzymes (e.g. Heinz bodies test).
Tests for Shortened Red Cell Lifespan:
A shortened red cell survival is best tested by 51Cr labelling method.
Normal RBC lifespan of 120 days is shortened to 20-40 days in moderate haemolysis and to 5-20 days in severe
haemolysis.
 ACQUIRED HAEMOLYTIC ANAEMIAS

 Acquired haemolytic anaemias are caused by a variety of extrinsic factors, namely: antibody

(immunohaemolytic anaemia), mechanical factors (microangiopathic haemolytic anaemia), direct toxic

effect (in malaria, clostridial infection etc), splenomegaly, and certain acquired membrane
abnormalities (paroxysmal nocturnal haemoglobinuria).
 IMMUNOHAEMOLYTIC ANAEMIAS
AUTOIMMUNE HAEMOLYTIC ANAEMIA (AIHA)

LABORATORY FINDINGS:
 Mild to moderate chronic anaemia.
 Reticulocytosis.
 Prominent spherocytosis in the peripheral blood film.
 Positive direct Coombs’ (antiglobulin) test for presence of warm antibodies on the red cell, best detected at 37°C.
 A positive indirect Coombs’ (antiglobulin) test at 37°C may indicate presence of large quantities of warm
antibodies in the serum.
 Unconjugated (indirect) hyperbilirubinaemia.
 Co-existent immune thrombocytopenia along with occasional venous thrombosis may be present (termed Evans’
syndrome).
 In more severe cases, haemoglobinaemia and haemoglobinuria may be present.
 ‘COLD’ ANTIBODY AIHA

LABORATORY FINDINGS:
 Chronic anaemia.
 Low reticulocyte count since young red cells are affected more.
 Spherocytosis is less marked.
 Positive direct Coombs’ test for detection of C3 on the red cell surface but IgM responsible for C3
coating on red cells is not found.
 The cold antibody titre is very high at 4°C and very low at 37°C (Donath-Landsteiner test). IgM class
cold antibody has specificity for I antigen, while the rare IgG class antibody of PCH has P blood group
antigen specificity.
 PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA (PNH)

i) Haemolytic anaemia.
ii) Pancytopenia (mild granulocytopenia and thrombocytopenia frequent).
iii) Intermittent clinical haemoglobinuria; acute haemolytic episodes occur at night identified by passage
of brown urine in the morning.
iv) Haemosiderinuria very common.
v) Venous thrombosis as a common complication.
The presence of inordinate sensitivity of red blood cells, leucocytes and platelets to complement in PNH
can be demonstrated in vitro by Ham’s test using red cell lysis at acidic pH or by sucrose haemolysis test.
About 20% cases of PNH may develop myeloproliferative or myelodysplastic disorder and some even
develop acute myeloid leukaemia.
 HEREDITARY SPHEROCYTOSIS

 Anaemia of mild to moderate degree.

 Reticulocytosis, usually 5-20%.
 Blood film shows the characteristic abnormality of erythrocytes in the form of microspherocytes
 MCV is usually normal or slightly decreased but MCHC is increased.
 Osmotic fragility test is helpful in testing the spheroidal nature of red cells which lyse more readily in
solutions of low salt concentration i.e. osmotic fragility is increased.
 Autohaemolysis test is similar to osmotic fragility test after incubation and shows increased
spontaneous autohaemolysis (10-15% red cells) as compared to normal red cells (less than 4%).
 Direct Coombs’ (antiglobulin) test is negative so as to distinguish this condition from acquired
spherocytosis of AIHA in which case it is positive. Spherocytes may also be seen in blood film in
acquired immune haemolytic anaemia and following red cell transfusion.
 RED CELL ENZYME DEFECTS (ENZYMOPATHIES)
G6PD DEFICIENCY

 During the period of acute haemolysis, there is rapid fall in haematocrit by 25-30%, features of
intravascular haemolysis such as rise in plasma haemoglobin, haemoglobinuria, rise in unconjugated
bilirubin and fall in plasma haptoglobin. Formation of Heinz bodies is visualised by means of
supravital stains such as crystal violet, also called Heinz body haemolytic anaemia. However, Heinz
bodies are not seen after the first one or two days since they are removed by the spleen, leading to
formation of ‘bite cells’ and fragmented red cells.
 Between the crises, the affected patient generally has no anaemia. The red cell survival is, however,
shortened. The diagnosis of G6PD enzyme deficiency is made by one of the screening tests (e.g.
methaemoglobin reduction test or MRT, fluorescent screening test, ascorbate cyanide screening test),
or by direct enzyme assay on red cells.
 PK DEFICIENCY
PYRUVATE KINASE (PK)

1. Normocytic and normochromic anaemia.

2. Reticulocytosis.
3. Blood film shows bizarre red cells.
4. Osmotic fragility is usually normal but after incubation it is increased.
5. Autohaemolysis is increased, but unlike hereditary spherocytosis, is not corrected by addition of
glucose.
6. Direct specific enzyme assay on red cells is the only method of establishing the diagnosis.
MISCELLANEOUS
 POLYCYTHAEMIA VERA

Polycythaemia vera (PV) is a clonal disorder characterized by increased production of all myeloid
elements resulting in pancytosis (i.e increased red cells, granulocytes, platelets) in the absence of any
recognizable cause.
PV IS DIAGNOSED BY THE FOLLOWING HAEMATOLOGIC FINDINGS:

1. Raised haemoglobin concentration (above 17.5 g/dl in males and 15.5 g/dl in females).
2. Erythrocytosis (above 6 million/µl in males and 5.5 million/µl in females).
3. Haematocrit (PCV) above 55% in males and above 47% in females.
4. Mild to moderate leucocytosis (15,000-25,000/µl) with basophilia and raised neutrophil alkaline
phosphatase scores.
5. Thrombocytosis with defective platelet function.
6. Bone marrow examination reveals erythroid hyperplasia or panhyperplasia.
7. Cytogenetic abnormalities such as 20q, trisomy 8 and 9p are found in 30% cases of PV.
8. In PV, unlike secondary polycythaemia, erythropoietin levels in serum and urine are reduced.
 HAEMOLYTIC DISEASE OF NEWBORN

 Haemolytic disease of the newborn (HDN) results from the passage of IgG antibodies from the

maternal circulation across the placenta into the circulation of the foetal red cells.

 Besides pregnancy, sensitisation of the mother may result from previous abortions and previous blood

transfusion. HDN can occur from incompatibility of ABO or Rh blood group system.

 ABO incompatibility is much more common but the HDN in such cases is usually mild, while Rh-D

incompatibility results in more severe form of the HDN.

 LABORATORY FINDINGS

The haematologic findings in cord blood and mother’s blood are as under:

1. Cord blood shows variable degree of anaemia, reticulocytosis, elevated serum bilirubin and a positive
direct Coombs’ test if the cord blood is Rh-D positive.

2. Mother’s blood is Rh-D negative with high plasma titre of anti-D.

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